Difference between revisions of "Team:BGU Israel/Demonstrate"
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<p class="justified">We wished to confirm that we could induce apoptosis in reactive astrocytes by expressing rev-Caspase3. As transfection efficiency of the astrocyte cell-line with our designed constructs had very low efficiency. We have consulted with Dr. Dinorah Friedmann-Morvinski from Tel-Aviv University who advised us that astrocyte are easier to infect with lenti-viral vectors rather than using other transfection methods. Hence, we decided to use infection by a lenti-viral vector as our delivery system.</p> | <p class="justified">We wished to confirm that we could induce apoptosis in reactive astrocytes by expressing rev-Caspase3. As transfection efficiency of the astrocyte cell-line with our designed constructs had very low efficiency. We have consulted with Dr. Dinorah Friedmann-Morvinski from Tel-Aviv University who advised us that astrocyte are easier to infect with lenti-viral vectors rather than using other transfection methods. Hence, we decided to use infection by a lenti-viral vector as our delivery system.</p> | ||
| − | <p>First, we wanted to demonstrate the ability of our system to work with human embryonic kidney 239 cells (HEK293) through the expression of a fluorescent marker (rather than inducing apoptosis). We have chosen to validate our system in HEK293 cells, which are much easier to transfect, infect and handle<sup>15,16</sup>. HEK293 cells were co-transfected with the similar plasmid that was designed and synthesized for the astrocyte-pathway. | + | <p>First, we wanted to demonstrate the ability of our system to work with human embryonic kidney 239 cells (HEK293) through the expression of a fluorescent marker (rather than inducing apoptosis). We have chosen to validate our system in HEK293 cells, which are much easier to transfect, infect and handle<sup>15,16</sup>. HEK293 cells were co-transfected with the similar plasmid that was designed and synthesized for the astrocyte-pathway.</p> |
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| − | pSynt-CMV contains a gRNA and the rev-caspase3 conjugated to mCherry under a synthetic promoter. The transfected HEK293 cells displayed mCherry fluorescence! This served as a proof of concept for the activity and the validity of our device. | + | For the HEK293 we used the generic CMV promoter for the regulation of expression of dCas9-VP64 and the gRNA rather than our specific reactive astrocyte promoters (lenti-viral-vector: CMV-dCas9-VP64 and pSynt-CMV). |
| + | pSynt-CMV contains a gRNA and the rev-caspase3 conjugated to mCherry under a synthetic promoter. The transfected HEK293 cells displayed mCherry fluorescence! This served as a proof of concept for the activity and the validity of our device.<br /> During the last week before the iGEM Jamboree, we not only saw mCherry in our C8D30 reactive astrocytes, but the mCherry was expressed under our specific promoters pSteap4 and pTimp1<br /> This indicates that when both the lenti-viral-vector:pTimp1-dCas9-VP64 and the pSynt plasmids are activated, the rev-Caspase3, which is conjugated to mCherry, is successfully expressed in our target cells!</p> | ||
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Revision as of 15:07, 17 October 2018
Demonstrate
Our hypothesis states that removing reactive astrocytes from the central nervous system (CNS) will slow the rate of neuron degeneration in reactive gliosis conditions. The proposed therapeutic approach has two main objectives: remove existing reactive astrocytes while simultaneously preventing the creation of new reactive astrocytes. We wish to achieve these objectives by inducing apoptosis in reactive astrocytes and inhibiting the secretion of pro-inflammatory cytokines by microglia.
A critical aspect of our entire system is specificity, in order to prevent damage to healthy astrocytes and to improve the treatment efficiency. We decided to increase specificity in three ways: specific promoters, two-plasmid system, and specific delivery to our target cells.
Astrocyte Activation
Our hypothesis relies on recent studies which show that, in ALS, microglia are activated by inflammation and secrete cytokines which transform resting astrocytes to reactive and toxic astrocytes. These reactive astrocytes then accelerate motor neuron death1,2. This entire mechanism is termed reactive gliosis3. First, we had to confirm that it is possible to model reactive gliosis in our chosen cell lines; BV2 microglia and C8D30 astrocytes. We chose to reproduce inflammation using lipopolysaccharide (LPS) which has been shown to model an infectious state4. LPS is a common immunogen used widely to activate the TLR4 pathway but also in ALS specific research1,3.
We used ELISA and Western Blot analysis in order to demonstrate an increase in a reactive astrocyte marker, the C3 protein5. The results of the ELISA show the highest expression of C3 protein when the astrocytes were treated with microglia conditioned medium that was previously activated by LPS, when compared to negative controls where astrocytes were not treated. The same was confirmed and demonstrated by the addition of commercial purified cytokines IL1a, TNFa, and c1q directly to astrocytes. These cytokines are commonly associated with activated microglia in ALS models6,7. These results have confirmed our hypothesis, that the LPS activated BV2 cell-line secretes cytokines which in turn activate the astrocytes. The Western Blot results were not significant.
However, surprisingly, we found that astrocytes which we had not activated expressed some C3 as well. Our immunoblot results confirmed that all our samples expressed C3 to some extent.
A consultation with Dr. Dinorah Friedmann-Morvinski from Tel-Aviv University taught us that astrocyte cell-lines are always reactive to a certain extent, since the cells are under stress when cultured in vitro, and resting astrocytes under stress conditions can turn to reactive astrocytes. This information has verified our findings so far. An immunostaining assay, performed by Dr. Friedmann-Morvinski’s lab on our astrocyte cell cultures using the markers GFAP and Nestin, confirmed the cell lines’ reactivity8. At this point we were confident that we had a reliable reactive gliosis model. Unfortunately, we understood that we did not have a resting astrocyte model to compare to
Cytokine Inhibition
We were able to synthesize 5 variants of this plasmid with different guide RNAs. However, several attempts to transfect our microglia cell-line with these plasmids were unsuccessful, and due to time limitations, we have slightly changed course. In order to achieve a proof of concept, we used a different plasmid delivered by a lentivirus vector which preformed RNAi knockdown to the IKKβ gene.
Following the IKKβ knockdown, we wished to demonstrate that an inhibition of cytokine expression can be achieved through targeting the NFΚβ pathway, a well-known and characterized inflammation pathway. The most promising strategy was to reduce the expression of IΚKB, which is a central protein in the NFkB pathway9.
We were able to reduce endogenous IΚKβ expression by infection of microglia BV2 cells with a lentiviral vector that introduces RNA interference10,11. The IKKß knockdown was confirmed through quantitative PCR as well as ELISA that have shown a reduction in TNFα cytokine (a cytokine expressed through the NfkB pathway) levels in BV2 microglia cells transfected with the relevant plasmid.
Reactive Astrocyte Specificity
A critical aspect in our project is to ensure that our system could be safe for human consumption by confirming that our system targets only reactive astrocytes and not healthy, resting astrocytes.
Our strategy is based on using specific promoters to drive expression of targeted genes only in reactive astrocytes and not in healthy ones. Therefore, we identified two promoters, Timp1 and Steap4, that should be active only in reactive astrocytes3,12-14.
These promoters were chosen, as these genes (according to the literature) have a much higher expression in reactive astrocytes when compared to resting astrocytes and other CNS cells3.
We performed a promoter assay and found out that indeed these promoters drove high expression of the reporter gene in C8D30, the cell line which serves as a model for reactive astrocytes.
As we did not have a resting astrocyte model, we could not test the activity of these promoters in resting astrocytes. However, based on available literature these genes are expressed in negligible amounts in resting astrocytes12.
Rev-Capase3 Induction
We wished to confirm that we could induce apoptosis in reactive astrocytes by expressing rev-Caspase3. As transfection efficiency of the astrocyte cell-line with our designed constructs had very low efficiency. We have consulted with Dr. Dinorah Friedmann-Morvinski from Tel-Aviv University who advised us that astrocyte are easier to infect with lenti-viral vectors rather than using other transfection methods. Hence, we decided to use infection by a lenti-viral vector as our delivery system.
First, we wanted to demonstrate the ability of our system to work with human embryonic kidney 239 cells (HEK293) through the expression of a fluorescent marker (rather than inducing apoptosis). We have chosen to validate our system in HEK293 cells, which are much easier to transfect, infect and handle15,16. HEK293 cells were co-transfected with the similar plasmid that was designed and synthesized for the astrocyte-pathway.
For the HEK293 we used the generic CMV promoter for the regulation of expression of dCas9-VP64 and the gRNA rather than our specific reactive astrocyte promoters (lenti-viral-vector: CMV-dCas9-VP64 and pSynt-CMV).
pSynt-CMV contains a gRNA and the rev-caspase3 conjugated to mCherry under a synthetic promoter. The transfected HEK293 cells displayed mCherry fluorescence! This served as a proof of concept for the activity and the validity of our device.
During the last week before the iGEM Jamboree, we not only saw mCherry in our C8D30 reactive astrocytes, but the mCherry was expressed under our specific promoters pSteap4 and pTimp1
This indicates that when both the lenti-viral-vector:pTimp1-dCas9-VP64 and the pSynt plasmids are activated, the rev-Caspase3, which is conjugated to mCherry, is successfully expressed in our target cells!
Final Remarks
These experiments demonstrate that our visionary two-component system indeed works in a reliable model. If time will allow, we would confirm apoptosis in the astrocyte cell lines and find a reliable resting astrocyte cell line model. Future work would test our system on primary astrocytes from ALS mouse models by using our designed plasmids with a cell-specific adeno-associated virus (AAV) delivery system and finally in live mouse models of ALS.
We considered the fact that our model is less ALS specific as a drawback, when in fact it can be viewed as an advantage. Our system is designed to be applicable to all ALS cases as well as to a broad range of diseases in which reactive gliosis appears, such as Huntington’s disease, Parkinson’s disease, brain injuries and brain cancers. Therefore, using a model which accurately portrays general reactive gliosis conditions, without mimicking only one specific disease, allows us to explore the application of our system on a wider range of diseases. We are very proud of the results we were able to achieve so far and are optimistic about the potential of our project to reach real world applications. Due to the novelty of our approach, and the therapeutic potential we envision for it, a provisional patent application has been submitted by our team.
References
- Liddelow, S. A., Guttenplan, K. A., Clarke, L. E., Bennett, F. C., Bohlen, C. J., Schirmer, L., … Barres, B. A. (2017). Neurotoxic reactive astrocytes are induced by activated microglia. Nature, 541(7638), 481–487.
- Haidet-Phillips, A. M., Hester, M. E., Miranda, C. J., Meyer, K., Braun, L., Frakes, A., … Kaspar, B. K. (2011). Astrocytes from Familial and Sporadic ALS Patients are Toxic to Motor Neurons. Nature Biotechnology, 29(9), 824–828.
- Zamanian, J., Xu, L., Foo, L., Nouri, N., Zhou, L., Giffard, R., & Barres, B. (2012). Genomic Analysis of Reactive Astrogliosis. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience , 32(18), 6391–6410.
- Alexander, C., Rietschel E. T. (2001) Bacterial lipopolysaccharides and innate immunity. Journal of Endotoxin Research, vol. 7, no. 3, pp. 167–202.
- Liddelow, S. A., Barres, B. A. (2017). Reactive Astrocytes: Production, Function, and Therapeutic Potential. Immunity, Volume 46, Issue 6, pp. 957 – 967.
- Brites, D., & Vaz, A. R. (2014). Microglia centered pathogenesis in ALS: insights in cell interconnectivity. Frontiers in Cellular Neuroscience, 8, 117.
- Geloso, M. C., Corvino, V., Marchese, E., Serrano, A., Michetti, F., & D’Ambrosi, N. (2017). The Dual Role of Microglia in ALS: Mechanisms and Therapeutic Approaches. Frontiers in Aging Neuroscience, 9, 242.
- Cho, J. M., Shin, Y.-J., Park, J.-M., Kim, J., & Lee, M.-Y. (2013). Characterization of nestin expression in astrocytes in the rat hippocampal CA1 region following transient forebrain ischemia. Anatomy & Cell Biology, 46(2), 131–140.
- Israël, A. (2010). The IKK Complex, a Central Regulator of NF-κB Activation. Cold Spring Harbor Perspectives in Biology, 2 (3), a000158.
- Wang, X., Wang, H., Yang, H., Li, J., Cai, Q., Shapiro, I. M., & Risbud, M. V. (2014). Tumor Necrosis Factor-α– and Interleukin-1β–Dependent Matrix Metalloproteinase-3 Expression in Nucleus Pulposus Cells Requires Cooperative Signaling via Syndecan 4 and Mitogen-Activated Protein Kinase–NF-κB Axis: Implications in Inflammatory Disc Disease. The American Journal of Pathology, 184(9), 2560–2572.
- Van den Haute, C., Eggermont, K., Nuttin, B., Debyser, Z., Baekelandt, V. (2003). Lentiviral Vector-Mediated Delivery of Short Hairpin RNA Results in Persistent Knockdown of Gene Expression in Mouse. Human Gene Therapy, vol. 14, No. 18, pp. 1799-1807.
- Zhang, Y., Chen, K., Sloan, S. A., Bennett, M. L., Scholze, A. R., O’Keeffe, S., … Wu, J. Q. (2014). An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and Vascular Cells of the Cerebral Cortex. The Journal of Neuroscience, 34(36), 11929-11947.
- Tokuda, E. , Okawa, E. and Ono, S. (2009), Dysregulation of intracellular copper trafficking pathway in a mouse model of mutant copper/zinc superoxide dismutase‐linked familial amyotrophic lateral sclerosis. Journal of Neurochemistry, 111: 181-191.
- Lorenzl, S., Albers, D. S., LeWitt, P. A., Chirichigno, J. W., Hilgenberg, S. L., Cudkowicz, M. E., Beal, M. F. (2003). Tissue inhibitors of matrix metalloproteinases are elevated in cerebrospinal fluid of neurodegenerative diseases. Journal of the Neurological Sciences , vol. 207 , Issue 1 , pp. 71-76.
- Mao, W.P, Ye, J.L, Guan, Z.B, Zhao, J.M, Zhang, C., Zhang, N., Jiang, P., Tian, T. (2007). Cadmium induces apoptosis in human embryonic kidney (HEK) 293 cells by caspase-dependent and -independent pathways acting on mitochondria, Toxicology in Vitro, vol. 21, Issue 3, pp. 343-354.
- Portier, B.P, Taglialatela, G. (2006). Bcl-2 localized at the nuclear compartment induces apoptosis after transient overexpression. The journal of biological chemistry, vol. 281, issue 52, pp. 40493-40502.